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A Novel On-Chip Method for Differential Extraction of

Sperm in Forensic Cases
Fatih Inci, Mehmet O. Ozen, Yeseren Saylan, Morteza Miansari, Duygu Cimen,
Raghu Dhara, Thiruppathiraja Chinnasamy, Mehmet Yuksekkaya, Chiara Filippini,
Deepan Kishore Kumar, Semih Calamak, Yusuf Yesil, Naside Gozde Durmus,
George Duncan, Leonard Klevan,* and Utkan Demirci*

sexual assault occurs in every 98 s in the

One out of every six American women has been the victim of a sexual assault United States alone, with the majority
in their lifetime. However, the DNA casework backlog continues to increase of victims being under the age of 30.[1–3]
outpacing the nation’s capacity since DNA evidence processing in sexual An investigative report in 2015 identi-
assault casework remains a bottleneck due to laborious and time-consuming fied over 70 000 sexual assault kits from
over 1000 police departments (≈6% of
differential extraction of victim’s and perpetrator’s cells. Additionally, a sig-
the police departments in the USA) that
nificant amount (60–90%) of male DNA evidence may be lost with existing were not tested for DNA evidence.[4]
procedures. Here, a microfluidic method is developed that selectively cap- Therefore, the demand for DNA testing
tures sperm using a unique oligosaccharide sequence (Sialyl-LewisX), a major is increasing. Expanded awareness of the
carbohydrate ligand for sperm-egg binding. This method is validated with power of forensic technology to help in
solving crimes creates new needs for sci-
forensic mock samples dating back to 2003, resulting in 70–92% sperm cap-
entific advances in the field.[5,6] Among
ture efficiency and a 60–92% reduction in epithelial fraction. Captured sperm these advances, microfluidic technologies
are then lysed on-chip and sperm DNA is isolated. This method reduces have considerable impact by combining
assay-time from 8 h to 80 min, providing an inexpensive alternative to current high-throughput processing and efficient
differential extraction techniques, accelerating identification of suspects and isolation of cells and biological entities
advancing public safety. from complex heterogeneous biological
matrices.[7–9]
In practice, processing of evidence
from sexual assault kits generally requires separation of the
1. Introduction
victim’s cells from the perpetrator’s cells. This process involves
The failure to test and analyze evidence connected to sexual time-consuming, labor-intensive steps of selective cell lysis,
assault in a timely manner constitutes a growing problem for centrifugation, and separation into female and male cell frac-
victims, public safety, and the criminal justice system. The tions (i.e., differential extraction) which can take up to 8 h,
Rape, Abuse & Incest National Network has reported that a contributing significantly to the backlog problem. However,

Dr. F. Inci, Dr. M. O. Ozen, Dr. Y. Saylan, Dr. M. Miansari, Dr. N. G. Durmus
Dr. D. Cimen, R. Dhara, Dr. T. Chinnasamy, S. Calamak, Department of Biochemistry
Y. Yesil, Prof. U. Demirci Stanford University
Bio-Acoustic MEMS in Medicine (BAMM) Laboratory Stanford Genome Technology Center
Canary Center at Stanford for Cancer Early Detection Palo Alto, CA 94304, USA
Department of Radiology Dr. G. Duncan
Stanford School of Medicine Crime Laboratory
Stanford University Broward County Sheriff’s Office
Palo Alto, CA 94304, USA Fort Lauderdale, FL 33301, USA
E-mail: [email protected]
Dr. L. Klevan
Dr. M. Yuksekkaya, C. Filippini, D. K. Kumar DxNow Inc.
Department of Medicine Gaithersburg, MD 20879, USA
Brigham and Women’s Hospital E-mail: [email protected]
Harvard Medical School
Prof. U. Demirci
Boston, MA 02115, USA
Department of Electrical Engineering (by courtesy)
© 2018 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Stanford University
Weinheim. This is an open access article under the terms of the Creative Stanford, CA 94305, USA
Commons Attribution License, which permits use, distribution and
reproduction in any medium, provided the original work is properly cited.

DOI: 10.1002/advs.201800121

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Figure 1. Workflow of on-chip differential extraction. i) In practice, samples are collected using a swab or cotton gauze in a forensic scene, where a
mixture of semen and epithelial cells are majorly present on the victim’s body and/or garments at the crime scene. ii) After collection, samples are
simply introduced into the device using single-step pipetting and incubated for an hour at room temperature. The channels are then washed and sperm
cells are specifically captured, while epithelial cells are removed due to their larger size and lack of an adhesion molecule on the channel surface. iii) The
captured sperm are treated with a lysis buffer on-chip, and sperm DNA is collected into a tube for potential forensic downstream genomic analyses.

it has been reported that this cell separation process results pellucida (ZP)) of oocyte, as a capture agent (Figure S1, Sup-
in losses of 60–90% of the male DNA.[10–13] Although there porting Information). This oligosaccharide sequence has been
have been multiple attempts for alternative methods to dif- reported as a major contributing element for human sperm-
ferentially extract sperm using acoustic trapping,[14] antibody- oocyte binding.[22,23] There are also components on the sperm
based capture,[15] laser microdissection,[16–18] nuclease-based membrane reported as docking units, including the β1–4
approaches,[19] and magnetic bead-based separation,[20,21] these galactosyltransferase 1 (B4GAL-T1) peripheral protein, which
methods have not been broadly available in practical applica- plays a crucial role in human sperm-oocyte binding.[24–29] To
tions due to the complexity and low separation yield for sperm. understand the dynamics of SLeX binding to sperm surface, we
As a result, they are not widely in use in the community. Par- used B4GAL-T1 as a model docking/binding unit on the sperm
ticularly, the antibody-based extraction methods have difficul- membrane and computed a molecular docking simulation to
ties to work with aged samples due to the changes in the antigen discover the locations and energetics of binding (Figure 2). In
specificity of sperm over time. Hence, this challenge makes this process, as shown in Figure 2a, we first extracted molecular
them less capable to capture sperm, which decreases to ≈17% structure of SLeX in silico from a protein complex defined in
after 10 days, limiting their utility and applicability for forensic the Protein Data Bank (PDB ID: 3PVD).[30] We then extracted
samples.[21] To address these unmet challenges, we have devel- B4GAL-T1 from human M340H-beta-1,4-galactosyltrans-
oped a microfluidic method integrated with a bioinspired ferase-1 (M340H-B4GAL-T1, PDB ID: 4EE3) (Figure S2a, Sup-
oligosaccharide sequence for selective isolation, differential porting Information).[31] The results of the docking simulations
extraction, and quantitation of sperm from the forensic evi- of molecular surfaces of SLeX and B4GAL-T1 were computed
dence of heterogeneous cellular content in sexual assault kits and visualized using AutoDock Vina and Visual Molecular
(Figure 1). Here, we present a method that i) differentially iso- Dynamics (VMD). The docking analysis revealed seventeen
lates sperm and lyses them on-chip, and extracts sperm DNA for potential binding modes with at least nine different locations
downstream genetic analyses; ii) reduces the differen- on the B4GAL-T1 surface for SLeX binding (Figure 2b–d and
tial extraction time from 8 h to 80 min; iii) minimizes the Figure S2b–e, Supporting Information). This study revealed
need for manual labor; iv) increases capture efficiency of strong binding modes with affinity energies ranging from
immuno-based separation of sperm assays from ≈17%[21] to −9.0 to −11.6 kcal mol−1 (Figure 2c). We observed a binding
70–92%; and v) keeps this high efficiency for samples older hot-spot at the Location #2, where eight of the seventeen SLeX
than 15 years, representing a crucial direction to reduce the molecules were bound (Figure S2c and Video S1, Supporting
evidence backlog. Information). Experimentally, we also confirmed that SLeX
decorated microfluidic surfaces was able to capture sperm with
various morphologies, including normal, condensed acrosome,
2. Results and Discussion abnormal middle-piece, large head, double heads, double tails,
small head (pin-head), and tail-less (Figure 2d). Given that SLeX
2.1. Molecular Docking Study targets the sperm head, binding and capture of sperm was
independent of sperm morphology. Specifically, sperm without
A recent study identified an oligosaccharide (i.e., Sialyl-LewisX a tail were also captured with SLeX agent primarily interacting
(SLeX: [NeuAcα2-3Galß1-4(Fucα1-3)GlcNAc])) as a unique mole- with the sperm head.
cule that sperm uses to bind to the egg.[22,23] Although this study
did not define exact mechanisms of binding, it created a new
direction to bind sperm selectively to surfaces, circumventing 2.2. Evaluating Surface Characteristics and Sperm Capture
the degradation problem that is inherent to antibodies that focus Efficiency in Microchannels
on the sperm surface for immuno-separation purposes.
In the experiments, we utilized this bio-inspired material, To efficiently capture sperm in microchannels, we integrated
i.e., SLeX, which is located on the extracellular matrix (i.e., zona SLeX with a microfluidic technology. Briefly, we designed

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Figure 2. Evaluation of SLeX binding kinetics and binding locations on sperm head. a) SLeX structure was extracted from a protein complex defined
in the Protein Data Bank (PDB ID: 3PVD) and visualized in silico. Computational analysis revealed the molecular surface of the SLeX agent for
sperm binding using VMD’s built-in SURF tool. b) β1–4 galactosyltransferase 1 (B4GALT1) was extracted from human M340H-beta-1,4-galactosyl-
transferase-1 (M340H-B4GAL-T1, PDB ID: 4EE3) and visualized in silico. This enzyme-receptor on the sperm plasma membrane plays a key role in
sperm-egg binding. B4GAL-T1-SLeX interactions were then computed using AutoDock Vina, and the analyses revealed at least nine unique locations
for seventeen potential binding modes for SLeX binding to B4GALT1. c) At these docking sites, strong binding was observed with the affinity energies
ranging from −9.0 to −11.6 kcal mol−1. d) We further observed that SLeX molecules capture sperm cells with different morphologies (i.e., normal,
condensed acrosome, abnormal middle-piece, large head, double heads, double tails, small head, and tail-less) on-chip. These experimental findings
confirmed our results observed in silico, indicating that SLeX targets sperm head and its binding is independent of distinct sperm morphologies. Scale
bars (black lines) represent 10 µm.

microchannels that consist of three layers: i) poly(methyl meth- serum albumin (BSA) blocking, ii) SLeX concentration, and
acrylate) (PMMA) for formation of inlets and outlets, ii) double- iii) channel height (Figure 3a). We first examined the effect of
sided adhesive (DSA) for the formation of microchannels and the 4-ABAH concentrations (0.25 and 2 mg mL−1) on sperm cap-
assembly of one PMMA and one glass layer, and iii) glass coverslip ture efficiency, keeping the SLeX concentration (0.1 mg mL−1)
surface (Figure S3, Supporting Information). Layer-by-layer, phys- and microchannel height (50 µm) constant. We observed
ical and chemical modifications are applied to the glass surface to higher capture efficiencies at 0.25 mg mL−1 of 4-ABAH concen-
immobilize SLeX (Figure S4, Supporting Information). tration but it was not statistically different (n = 3–4, p > 0.05)
Capture efficiency was assessed by varying three main (Figure 3b). Further, this may point to a potential steric hindrance
parameters: i) concentration of mediator agent (i.e., 4-amin- in higher mediator concentrations for SLeX immobilization. As
obenzoic acid hydrazide: 4-ABAH) and evaluation of bovine reported in the literature, more densely packed layers revealed

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Figure 3. Evaluation of surface chemistry and microfluidic chip parameters for sperm capture. a) Glass surfaces were decorated with SLeX agent
using a layer-by-layer surface chemistry approach. Capture efficiency was evaluated by varying three parameters: i) concentration of mediator molecule
(i.e., 4-Aminobenzoic acid hydrazide: 4-ABAH) and bovine serum albumin (BSA), ii) SLeX concentration, and iii) channel height. b) Various 4-ABAH
(0.25 mg mL−1 and 2 mg mL−1) and BSA concentrations (0% and 3%) were examined, and sperm capture efficiency was calculated at each concentra-
tion. In these experiments, 50 µm high microchannels were modified with a fixed SLeX concentration (0.1 mg mL−1). Here, 0.25 mg mL−1 of 4-ABAH
provided higher capture efficiency than 2 mg mL−1 of 4-ABAH but it was not statistically different (n = 3–4, p > 0.05). Further, this might be due
to potential steric hindrance for SLeX immobilization to the surface. Further, BSA blocking did not significantly affect the sperm capture efficiency
(n = 3–4, p > 0.05) in these experimental sets. c) Different SLeX concentrations ranging from 0.1 to 0.5 mg mL−1 were used to evaluate sperm cap-
ture. The 50 µm high microchannels were modified with the optimized 4-ABAH (0.25 mg mL−1) and BSA (3%) concentrations. Here, 0.5 mg mL−1
of SLeX concentration provided higher capture efficiency compared to the other groups. Further, the capture efficiency in 0.5 mg mL−1 of SLeX was
not statistically different than the other SLeX concentrations (n = 4, p > 0.05). d) Two channel heights (50 µm and 80 µm) were evaluated in terms
of sperm capture efficiency. The microchannels were decorated with the optimized 4-ABAH (0.25 mg mL−1), BSA (3%), and SLeX (0.5 mg mL−1)

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lower surface activity.[32,33] This effect also indicated the link trying to move, they were also stuck to the channel surface due
between surface coverage, immobilization of molecules and to high capture capacity of SLeX material (Video S2, Supporting
capture activities, and therefore, a lower density of immobi- Information).
lization process on the surface provided a higher binding and To confirm surface functionalization and SLeX binding,
sensitivity.[32,34] Since 4-ABAH has a role to immobilize SLeX we performed Fourier Transform Infrared-Attenuated Total
molecules in the next step, much lower concentrations of Reflectance (FTIR-ATR) measurements on the modified micro-
4-ABAH will potentially not provide more binding points for channels (Figure 3e). As the fingerprint region of SLeX was
binding of SLeX agent to the surface. Therefore, we determined designated between 900 and 1280 cm−1,[35] we first analyzed this
to use 0.25 mg mL−1 of 4-ABAH in the experiments. In addi- range and observed CH wagging at 962 cm−1, CO stretching/
tion, we utilized BSA as a blocking agent, which has ideally dual bending at 1025 and 1082 cm−1, CO stretching at 1134 cm−1,
roles: i) blocking agent blocks nonmodified spots in the micro- and CN stretching at 1181 cm−1. These absorbance spectra
channel to minimize nonspecific binding of the other cells/ values appear to be shifted from unbound SLeX molecule,[35]
biological entities (e.g., epithelial cells); and ii) it does not signif- which might be caused by the generation of chemical bonding
icantly affect sperm capture while minimizing the nonspecific during immobilization process. Since glass has intense char-
binding. In the experiments, BSA blocking did not significantly acteristic spectrum between ≈682 cm−1 and ≈1200 cm−1 (e.g.,
change sperm capture efficiency (n = 3–4, p > 0.05). To mini- SiOH bending and SiOSi stretching),[36–38] the signal inten-
mize nonspecific binding, we also utilized BSA in the specificity sities of peaks in this region reduced. Further, the absorption
experiments to capture sperm from complex heterogeneous peak around 3500 cm−1 represented OH stretching vibrations
cell population including epithelial cells. This experimental set due to high number of free OH groups on SLeX mole­cule.
achieved a 76.5 ± 6.0% of capture efficiency when 0.25 mg mL−1 The intensity band appearing around 2850–2950, 2500–3000,
of 4-ABAH and 3% of BSA were applied with the other constant 1733, and 1630–1690 cm−1 were caused by CH bending, OH
parameters of SLeX and channel height. Next, we evaluated the stretching, CO (ester) stretching, and CO (amide) stretching
effect of SLeX concentrations varying from 0.1 to 0.5 mg mL−1 vibrations of SLeX molecule, respectively.[39] We further charac-
over sperm capture efficiency, keeping the microchannel height terized hydrophilicity of microchannel surface after the modi-
(50 µm), 4-ABAH (0.25 mg mL−1) concentration and BSA (3%) fication (Figure 3e-inset). The contact angle value of the bare
constant (Figure 3c). We observed that the increase in SLeX con- glass surface reduced from 48.5° ± 4.3 to 13.7° ± 3.1, indicating
centration enhanced sperm capture efficiency, and the highest that SLeX immobilization with layer-by-layer surface chemistry
SLeX concentration (0.5 mg mL−1) resulted in 86.1 ± 6.8% of generated more hydrophilic surface. These two different char-
capture efficiency by generating more binding sites for sperm acterization methods confirmed that SLeX molecule was suc-
capture. Further, this increase in capture efficiency was not statisti- cessfully immobilized to the microchannel surface.
cally different than the other SLeX concentrations (n = 4, p > 0.05).
Finally, we evaluated the effect of microchannel height on sperm
capture efficiency when we kept the aforementioned concentra- 2.3. Evaluating Distribution of Sperm Capture in Microchannels
tions (4-ABAH: 0.25 mg mL−1 and SLeX: 0.5 mg mL−1). Given
that increased surface interactions are vital for cell capture, we We assessed the spatial distribution of sperm on-chip by
observed higher capture efficiency with 50 µm high channel counting sperm before and after phosphate buffered saline
design compared to 80 µm high channel design (Figure 3d). (PBS) washing steps. In this experiment, we applied high
Overall, the highest sperm capture efficiency was achieved using and low sperm counts into the channels. During the imaging
i) 0.25 mg mL−1 of 4-ABAH and 3% BSA, ii) 0.5 mg mL−1 of studies, the entire channel was divided into 30 columns (hori-
SLeX, and iii) 50 µm high microchannel. We applied these zontal direction) by 10 rows (vertical direction). First, we evalu-
parameters to the following experimental designs to capture ated ≈8000 sperm per channel (high sperm count) (Figure 3f).
sperm. In the experiments, we also observed that sperm cells Before the washing step, we observed homogenous distribution
were tightly captured in microchannels. Although sperm were of sperm in a horizontal direction, whereas higher cell numbers

concentrations. We observed that 50 µm high channel heights resulted in higher capture efficiency than an 80 µm high channel. e) Surface functionaliza-
tion and SLeX binding on the modified channels were confirmed by Fourier Transform Infrared-Attenuated Total Reflectance (FTIR-ATR) measurements.
At the fingerprint region of SLeX (900 cm−1 to 1280 cm−1), we observed CH wagging at 962 cm−1, CO stretching/bending at 1025 and 1082 cm−1,
CO stretching at 1134 cm−1, and CN stretching at 1181 cm−1. Due to characteristic absorption region of glass between ≈682 cm−1 and ≈1200 cm−1
(e.g., SiOH bending and SiOSi stretching), the signal intensities of peaks in this region reduced. We also observed absorption peaks at 3500,
2850–2950, 2500–3000, 1733, and 1630–1690 cm−1 caused by OH stretching, CH bending, OH stretching, CO (ester) stretching, and CO
(amide) stretching vibrations of SLeX molecule, respectively. By performing contact angle measurements, we evaluated hydrophilicity properties after
surface modification (inset figure). The contact angle value of the bare glass surface altered from 48.5° ± 4.3 to 13.7° ± 3.1 after SLeX modification.
According to the FTIR-ATR and contact angle measurements, SLeX molecule was successfully immobilized to the microchannel surface. f) Spatial
distribution of cell capture was analyzed on-chip by imaging the entire microchannel surface through a tiling function of the microscope with an auto-
mated x–y stage. Sperm counts before and after the washing step were plotted through horizontal and vertical directions. Before the washing step, a
homogenous cell distribution was observed in a horizontal direction, whereas sperm cell count increased in the middle of the channels on the vertical
axis. The cell count was altered in the horizontal direction after the washing step and most of the sperm close to the inlet washed away from the channel
surface. On the other hand, the distribution trend at the vertical axis did not change after the washing step. For statistical analysis, we used one-way
ANOVA with Tukey’s post hoc test for multiple comparisons with the statistical significance threshold set at 0.05 (p < 0.05). Data is represented with
average value ± standard deviation (n = 3–4).

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were counted in the middle of the channel while scanning the 2.4. Benchmarking Nonspecific Cell Binding (Control)
vertical-axis. After the washing step, the cell count decreased in
the first 5–10 lanes close to the inlet in the horizontal direction. In control experiments, we did not decorate the channels with
On the other hand, the vertical distribution did not change after surface chemistry, and the glass surface was only cleaned
the washing step. In the second experimental set, we applied a with EtOH before being assembled (Figure 4). We then intro-
lower sperm count (≈300 sperm per channel) (Figure S5, Sup- duced 15 µL of samples with high and low sperm counts into
porting Information). Before the washing step, we observed the channels. High cell counts were defined as being between
nearly homogenous cell distribution in a horizontal direction. 750 and 1800 sperm per channel, whereas the low cell count
Through the vertical axis, we observed the same trend as with was around 100–300 sperm per channel. In high cell count
higher sperm count experiments, and the sperm cell count experiments, only a limited number of sperm remained
was higher in the middle of channel. After the washing step, (275 ± 96 cells) in the control surfaces without surface
the sperm count close to the inlet was altered in a horizontal chemistry when we applied 1742 ± 239 cells to the channels
direction, which was similarly observed in higher sperm count (Figure 4a). As a result, sperm samples with high cell counts
experiments. After washing, the vertical axis also had a similar were significantly removed from the channel surfaces in the
distribution trend, as observed before the washing step. absence of surface chemistry (n = 4, p < 0.05). In low cell count

Figure 4. Evaluation of nonspecific sperm cell binding (control) and limit of detection. a) The microchannels without surface chemistry were used
as a control set. Nonspecific sperm cell binding was assessed with high (750–1800 sperm per channel) and low (100–300 sperm per channel) cell
numbers. Only a limited number of sperm (275 ± 96 cells) remained in the channels when we applied 1742 ± 239 cells into the microchannels. Sperm
samples with a high cell number were significantly removed from the channel surfaces in the absence of surface chemistry (n = 4, p < 0.05). In addi-
tion, some sperm (186 ± 97 cells) remained when we introduced 285 ± 111 cells to the microchannels (n = 4, p > 0.05). These results demonstrated
that the bare glass surface itself has ≈200 nonspecific binding points over the sperm count range. b) We also evaluated the detection capability of
microchannels modified with surface chemistry. Most sperm (748 ± 9 cells) were captured when we applied 798 ± 9 cells into the microchannels
(n = 3, p > 0.05). In low cell count experiments, we observed that 116 ± 17 sperm were captured on-chip when we introduced 134 ± 19 cells to the
microchannels (n = 3, p > 0.05). As demonstrated in the plot, the microchannels modified with surface chemistry efficiently captured sperm in both
high and low cell numbers with ≈94% and ≈86% efficiency, respectively. c) Further, cell numbers were converted into percentage of sperm remaining
in microchannels after washing step. Higher ratios of sperm remained on the surface chemistry applied channels than that of control surfaces without
surface chemistry (n = 3–4, p < 0.05). We also observed that ≈200 bindings were mainly due to sperm-glass surface interactions in control surfaces
(n = 4, p < 0.05). In these experiments, we introduced 15 µL of samples with high and low sperm counts into the channels. d,e) We evaluated the limit
of detection parameter for the microchannels by applying multiple cell concentrations varying from ≈20 to ≈8000 cells per channel. The microchannels
captured down to ≈20 sperm cells per channel with a capture efficiency of 75.4 ± 1.5% (n = 3, p < 0.05), and the capture efficiency increased up to
93.6 ± 3.0% at higher cell counts (up to ≈8000 cells per channel), indicating that the microchannels were able to handle a broad range of cell numbers
and the capture capability of chips was independent of high cell numbers introduced into the microchannels. f) Limit of detection parameter was further
analyzed through a nonlinear fitting function. The curve had a linearity of 0.94 and 0.87 for R2 (Coefficient of determination: COD) and adjusted R2,
respectively. For statistical analysis, we used one-way ANOVA with Tukey’s post hoc test for multiple comparisons with the statistical significance
threshold set at 0.05 (p < 0.05). Horizontal brackets and asterics demonstrate statistically significant differences between groups. Data is represented
with average value ± standard deviation (n = 3–4).

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experiments, some sperm (186 ± 97 cells) remained when we of SLeX; and ii) washing step might be more effective over
introduced 285 ± 111 cells to microchannels without surface capture efficiency in samples with lower sperm counts since
chemistry (n = 4, p > 0.05) (Figure 4a). In control channels for sampling size varies by ≈20 – ≈8000 sperm per channel. This
both high and low cell count experiments, we observed that the could potentially contribute to changes in capture efficiency
bare glass surface itself had ≈200 nonspecific cell adherence parameter due to different effective ratio of sperm removed
points over all sperm count ranges introduced into the channel. from the surface after washing step. Further, in the experi-
After that, we further evaluated sperm counts in the channels ments, we observed a nonlinear trend with 0.94 and 0.87 for
modified with surface chemistry (Figure 4b). In high cell count R2 (Coefficient of determination: COD) and adjusted R2, respec-
experiments, most sperm (748 ± 9 cells) were captured when tively. The curve was also examined in two regions: i) low cell
we applied 798 ± 9 cells into microchannels (n = 3, p > 0.05). In count (≈20 to ≈300 sperm per channel), and ii) high cell count
low cell count experiments, 116 ± 17 sperm were captured in (≥ 300 sperm per channel). Samples lower than 300 sperm per
the channels when we introduced 134 ± 19 cells to the micro- channel range provided a capture efficiency between 75.4% and
channels (n = 3, p > 0.05). Comparing the data between surface 86.3%, whereas the capture efficiency for above 300 sperm per
chemistry applied channels and control surfaces (no surface channel reached up to 93.6 ± 3.0% (Figure 4f).
chemistry) in high cell count experiments, a high ratio of sperm
(≈94%) was captured on the surface chemistry decorated chan-
nels, whereas cells were significantly removed in control chan- 2.6. Evaluating Specificity of Sperm Capture in Microchannels
nels and only ≈16% of sperm remained in the control channels
(Figure 4b). We further analyzed the data and converted cell Vaginal samples in sexual assault kits typically contain vaginal
numbers into percentage of sperm remaining in microchannels epithelial cells from the victim and sperm cells from the per-
after washing step (Figure 4c). In both high and low cell count petrator. To evaluate specificity performance of microfluidic
experiments, greater ratios of sperm remained on the surface chips, we designed two experimental sets: i) microchannels
chemistry applied channels than that of control surfaces, indi- surfaces decorated with SLeX molecules, and ii) microchannels
cating that surface chemistry (SLeX coating) has significant surfaces modified up to the 4-ABAH binding step (non-SLeX).
effect on sperm capture efficiency for both low and high sperm In both experimental sets, we worked with a heterogeneous cell
concentrations applied to the channels (n = 3–4, p < 0.05). population including sperm and buccal epithelial cells. Thus,
Additionally, we observed the nonspecific cell adherence effect we evaluated whether SLeX is crucial in specific capture of
on the control surfaces, and ≈200 bindings were mainly due sperm from mixed cell populations (Figure 5). In these experi-
to sperm-glass surface interactions (n = 4, p < 0.05). These ments, the entire microchannel was scanned to count sperm
≈200 nonspecific binding points were consistent for both low and epithelial cells before and after washing steps (Figure S6,
and high concentrations applied to the channels, indicating the Supporting Information). On SLeX-modified surfaces, the
highest limit of nonspecific binding for the current channel percentage of captured sperm cells (≈91%) was statistically
size, geometry, and surface chemistry. Overall, the micro- greater than nonspecifically bound epithelial cells (≈7%) (n = 5,
channels modified with surface chemistry efficiently captured p < 0.05). Considering the necessity of SLeX to capture sperm,
sperm with a range of 86–94% in both high and low cell count we observed a drastic decrease in the percentage of captured
experiments. sperm on non-SLeX surfaces (n = 5, p < 0.05). No statistical
difference was observed in the percentage of remaining epi-
thelial cells in both non-SLeX and SLeX-coated channels
2.5. Evaluating Limit of Detection (LOD) (n = 5, p > 0.05). Overall, in these experiments, we obtained
two critical outcomes: i) SLeX-modified surfaces specifically
We assessed this parameter by applying multiple cell counts captured sperm and a vast majority of epithelial cells (≈93%)
(≈20 to ≈8000 sperm per channel) into the channels and calcu- were removed after a single wash step; and ii) SLeX played a
lating capture efficiency at each cell concentration (Figure 4d–f). pivotal role in capturing and isolating sperm from a heteroge-
As a result, the channels captured down to ≈20 sperm per neous cell population (Figure 5b,c and Figure S6, Supporting
channel with a capture efficiency of 75.4 ± 1.5%, and capture Information).
efficiency increased up to 93.6 ± 3.0% at higher cell counts (at
≈8000 sperm per channel) (Figure 4d,e). Therefore, the micro-
channels were able to handle a broad range of cell numbers and 2.7. Validating Microfluidic Chip Performance
the capture capability of microfluidic chips was independent with Forensic Mock Samples
of high cell counts introduced into the channels. Statistical
assessments demonstrated that capture efficiency derived from Forensic mock samples were collected from the Broward
≈20 sperm per channel experiment was lower than the other Sheriff’s Office Forensic Laboratory. In validation studies,
cell concentration groups (n = 3–9, p < 0.05), and also, there samples were sent to Stanford University under the approved
was no statistical difference between ≈35 and ≈8000 sperm per IRB protocol. The collected samples were noncasework/mock
channel (n = 3–9, p > 0.05) (Figure 4d,e). Since surface chem- samples, including epithelial cells and sperm. According to the
istry and channel parameters were all same, there might be guidelines of Broward Sheriff’s Office Forensic Laboratory, five
two reasons: i) the binding sites of SLeX in the microchannels mock samples from 2003 to 2015 were collected with either
might be saturated while applying samples with higher sperm cotton swab or cotton gauze, and directly introduced through
counts and this might have helped the capturing performance SLeX-decorated channels with three replicates (Figure 5d).

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Sperm cells were counted before and after washing steps. In ranging from ≈70% to ≈92% were observed for aged mock
the forensic mock samples, we observed a various number samples (Figure 5e,f). Additionally, as reported in the literature,
of sperm and epithelial cells. Sperm after washing step was cotton content interferes with capture performance of assays,[21]
counted in the microchannels, and high capture efficiencies and we observed similar hindrance when a large cotton swab

Figure 5. Specificity experiments and validation of microfluidic chips with forensic mock samples. a) Specificity of SLeX was tested with a heteroge-
neous cell population consisting of a male’s sperm and buccal epithelial cells collected from a female’s inner cheeks. Two sets of microfluidic chips were
prepared: i) all surface chemistry steps including SLeX and ii) all surface modifications without SLeX. b) SLeX-modified surfaces provided 91.1 ± 3.1%
of capture efficiency, whereas sperm cells drastically washed away from the surfaces without SLeX (n = 5, p < 0.05). In addition, SLeX provided high
specificity to capture sperm (≈91%) compared to epithelial cells (≈7% and ≈1%) in both experimental sets (n = 5, p < 0.05). There was no significant
binding of epithelial cells observed in the microchannels with SLeX and without SLeX (n = 5, p > 0.05). c) Microphotography was performed before and
after the washing steps on microchannels with SLeX. Black arrows represent epithelial cells (ECs) in the microchannels. Scale bars represent 50 µm.
d) Simulated forensic samples (noncasework samples) were obtained from the Broward Sheriff’s Office Forensic Laboratory. Five different mock
samples were introduced into the microchannels modified with SLeX, and the numbers of sperm were then counted before and after the wash steps.
We observed various numbers of sperm ranging from ≈300 to ≈745 cells, and most of the sperm cells were captured in the microchannels. e) Mock
samples provided high capture efficiencies, spanning from ≈70% to ≈92%. f) The mock samples were collected, using either cotton swab or cotton
gauze, on different dates and they consisted of different cell content and concentrations. The details were presented in the table. Data was represented
with average value ± standard deviation (n = 3). For statistical analysis, we used one-way ANOVA with Tukey’s post hoc test for multiple comparisons
with the statistical significance threshold set at 0.05 (p < 0.05). Horizontal brackets demonstrate statistically significant differences between groups.
Data is represented with average value ± standard deviation (n = 5).

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was used. For instance, in the FMS2 and FMS5, the capture over time since proteins on the sperm membrane aged over a
efficiency decreased to ≈82% and ≈70%, respectively. Whether long-term storage, as well as during drying process.[15,20,21] As
a full size of cotton swab or just a portion of cotton swab was we have shown in this study, SLeX has multiple binding sites
used, the capture efficiency ranged from 86% to 92% (FMS1 on the sperm surface, making it a unique element for aged
and FMS4). We also counted the retained epithelial cells in forensic sperm samples, allowing our methods to achieve
the channels and observed a significantly lower number of ≈5-fold higher sperm capture efficiency. Our technology solves
epithelial cells compared to the captured sperm count (n = 3, a significant problem that has failed to find a solution in the
p < 0.05) (Figure S7, Supporting Information). As demon- past for efficient differential extraction of sperm.
strated in the spiking experiments, we also confirmed that our Here, we integrated microfluidics with a unique oligosaccha-
microchannels were able to specifically capture sperm from a ride unit (i.e., SLeX), a major binding ligand for egg and sperm
heterogeneous cell population, and device performance was interaction. By introducing bioinspired materials into a micro-
not significantly changed while capturing sperm from aged fluidics realm, we have developed a powerful platform to selec-
forensic mock samples. tively isolate sperm in heterogeneous matrices by performing
only few steps (four sampling/washing and two incubation
steps) to provide on-chip sperm DNA lysate within 80 min. All
2.8. Sperm Lysis On-Chip and DNA Quantification sampling and extraction steps can be performed by existing
forensic DNA laboratory equipment and techniques such as
Captured sperm in microchannels were first treated with sample loading with a pipette and a single-flow rate wash for
tris(2-carboxyethyl)phosphine (TCEP) in Triton X-100 to lyse controlling selective removal of unbound cells from microchan-
cells on-chip. The collected lysate solution was then pro- nels. We validated this procedure with forensic mock samples
cessed through Proteinase K and spin column protocols, as shelved for over a decade, and we succeed to differentially
described in the Materials and Methods section. After these capture sperm cells in channels with high capture efficiency
protocols, the DNA concentration of each sample was meas- (70–92%).
ured and demonstrated in Table 1. Since each sperm cell This method is still in development, and we expect the fol-
includes ≈3 pg of DNA material, the captured cell number lowing improvements in the design and workflow. First, the
was then converted into an expected DNA concentration of current design of chips has up to 4 microchannels and process
each sample. We counted different number of sperm ranging 5–15 µL of sample volume per channel, which is typical in a
from 3160 to 7731 in the microchannels. After lysis step, we case sample. By integrating various designs of channel lateral
quantified DNA amount of collected samples within a range dimensions and numbers, the platform can potentially handle
from 79 to 188 pg µL−1 (Table 1). According to all these results, larger sample volumes for high-throughput DNA extraction.
we achieved sperm lysis on-chip and confirmed high DNA Second, as the incubation time for sperm capture takes 75% of
recovery with efficiency ratios between ≈52.8% and ≈88.6%, total processing time, this assay time would potentially be fur-
demonstrating the applicability of our platform for potential ther reduced by decreasing channel height. Third, although the
forensic downstream analyses. current system uses a simple hand-pipette and a syringe pump
in the sampling and washing steps, the entire platform can
potentially be automated by integrating an automatic pipetting
3. Conclusion system, as well as creating a closed-box system that minimizes
personnel integration and person-to-person variability. Also,
The differential extraction of sexual assault samples from automated preparation techniques using a robotic arm could
sexual assault kits requires up to 8 h of skilled personnel to considerably minimize potential batch-to-batch variations.
complete. Even while performing lengthy sample process steps, Fourth, in this study, sexual assault kit samples were visualized
a significant amount (60–90%) of male DNA may be lost during using a standard laboratory microscope. The next generation
existing procedures as reported in the literature.[10–13] Here, we of this platform can potentially be integrated with a portable
present a next-generation differential extraction technology that imaging system,[40] enabling easy access to the technology in
is, to the best of our knowledge, the most rapid, reliable, accu- remote locations for sexual assault evidence screening. Fifth, we
rate, user-friendly method available. Although there are previ- decorated channels with SLeX monomer units in the current
ously antibody-based capture approaches proposed for forensic study. Higher concentrations of SLeX (more than 0.5 mg mL−1)
samples, they suffer from loss of efficiency and specificity can also be utilized to evaluate its effect over sperm capture
efficiency. Ideally, a bioprinting strategy will further accelerate
Table 1. Efficiency of sperm lysis on-chip and quantification of lysed to increase the coverage rate of SLeX in the channels. Sixth,
sperm DNA. although the present platform utilizes affordable components
such as plastic layers, polymers, and glass slides, the cost of
Sample Sperm count Expected DNA concentration Qubit result Efficiency goods used for the fabrication and surface chemistry can poten-
ID on-chip [pg µL−1] [pg µL−1] (%) tially be reduced further with mass production.
S1 7731 ≈289.9 188 ≈64.8% Overall, the presented microfluidic technology with a bio-
S2 4990 ≈149.7 79 ≈52.8% inspired oligosaccharide sequence addresses critical technical
challenges in forensic rape cases, facilitating downstream
S3 5237 ≈159.8 91 ≈57%
genomic analyses, accelerating identification of suspects,
S4 3160 ≈94.8 84 ≈88.6%
and advancing public safety. In addition, the ability of our

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technology i) to differentially extract sperm from heterogeneous FTIR-ATR Spectroscopy: After surface chemistry was applied to the
cell population, ii) lyse sperm on-chip, and iii) extract sperm microchannels, the PMMA and DSA layers were removed for FTIR-ATR
measurements. The SLeX functionalized mircochannel was then placed
DNA within a short assay-time can open up new avenues for
on the FTIR-ATR instrument (Thermo Fisher Scientific, Nicolet iS10,
forensic downstream analyses. Waltham, MA, USA) and total light reflection was recorded from 650 to
4000 cm−1 range with 2 cm−1 of resolution.
Contact Angle Measurements: KRÜSS Drop Shape Analyzer (DSA100,
Hamburg, Germany) instrument was utilized for contact angle
4. Experimental Section measurements. The contact angle values were recorded with sessile
drop method by dropping 5 µL of ultrapure water and calculated as
Materials: (3-Mercaptopropyl)trimethoxysilane (3-MPS, 95%),
the average of the three different drops.
aminobenzoic acid hydrazide (4-ABAH, 95%), bovine serum albumin
Sampling and Counting: For spiked sperm samples, sperm were
(BSA), dimethyl sulfoxide (DMSO), Triton X-100, Proteinase K
purchased from California CryoBank under an Institutional Review Board
(recombinant, PCR Grade), and ethanol (EtOH, 200 proof) were
(Stanford University IRB Number: 6208, and Protocol ID: 30538). Frozen
purchased from Sigma-Aldrich (St. Louis, MO). N-γ-maleimidobutyryl-
sperm vials were briefly thawed in a water-bath set at 37 °C, and the
oxysuccinimide ester (GMBS), TCEP, and Qubit dsDNA High Sensitivity
numbers of sperm in each sample were counted using a hemocytometer.
(HS) Assay were obtained from Thermo Fisher Scientific (Waltham,
Before sampling, sperm were incubated at room temperature for 1–3 d.
MA). PBS and SLeX were obtained from Fisher Scientific (Hampton,
For sampling, 5–15 µL of sample was applied into the microchannels
NH), Zymo Research (Irvine, CA), and EMD Millipore (Hayward,
to ensure the channels filled with the sample. Sperm samples were
CA), respectively. QIAamp DNA Mini Kit was purchased from Qiagen
incubated for an hour while the imaging was being performed within
(Valencia, CA).
the entire microchannel using a tiling function of the light microscope
Molecular Docking Study: A molecular docking simulation is employed
with a motorized x–y stage (Zeiss, Germany) (before the washing step).
to study the binding localization and energy of SLeX – M340H-β-1,4-
The microchannels were then washed with 1xPBS for 20 min using a
galactosyltransferase-1 (M340H-B4GAL-T1 (B4GAL-T1) interactions on
syringe pump with a 5 µL min−1 flow rate to remove unbound cells. The
sperm membrane. The structural coordinate data of SLeX and B4GAL-T1
captured cells within the microchannels were counted (after the washing
were extracted from the Protein Data Bank.[30,31] The molecular surfaces
step). A second imaging step was performed to count the number of
of SLeX and B4GAL-T1, along with the results of the docking simulations,
captured sperm on-chip. Sperm counts before and after the washing
were computed and visualized using VMD.[41] AutoDock Tools (ADT)
steps were manually calculated using these microscope images. The
4.2 was utilized to configure the simulation input files.[42] SLeX and
capture efficiency rate was defined as (Equation 1)
B4GAL-T1 were converted into the PDBQT file format. AutoDock Vina
was then used for the molecular docking simulation,[43] followed by
Sperm count after washing
another ADT run to assess ligand-receptor hydrogen bonding and Capture Efficiency ( % ) = × 100  (1)
binding affinities. Binding affinities were reported as −kcal mol−1 for Sperm count before washing
each interaction.
Microchannel Fabrication: The microfluidic chips consisted of three In specificity experiments, buccal epithelial cells were collected from
main components: i) a PMMA layer (3.2 mm of thickness), ii) a DSA film female individuals and were mixed with sperm samples. The specificity
(50 and 80 µm of thickness), and iii) a glass cover slide (24 × 40 mm). experiments also followed the same sampling procedure as described
Versa LASER (Universal Laser Systems Inc., Scottsdale, AZ) and above.
CorelDRAW software (Ottawa, Ontario, Canada) were utilized to design Forensic Mock Samples: Simulated forensic samples were prepared
and cut PMMA layers and DSA films. Inlets and outlets of the chips by members of the Broward Sheriff’s Office Crime Laboratory (not
(0.65 mm in diameter, 26 mm apart) were milled into a PMMA layer, from casework evidence). Cuttings (cotton swab or cotton gauze)
and the DSA film provided microfluidic channels. The microfluidic chips from these samples were eluted in 500 µL of 1 × PBS and placed in
were then constructed by assembling these three components. Glass a 4 °C Thermomixer (Eppendorf, Germany) that was set at 1000 rpm
cover slides were used as a substrate material, where we performed for approximately an hour. The cuttings were removed and placed in
surface chemistry for sperm capture. spin baskets that were subsequently centrifuged for 5 min at
Surface Functionalization: Glass cover slides were first cleaned 16 100 rcf/13 200 rpm to pellet the solids in the solution. Afterward,
with absolute EtOH (200 proof) via sonication for 15 min at room ≈300 µL of the 1 × PBS was removed without disturbing the pellet.
temperature. The slides were immediately dried under either N2 gas or The pellet was resuspended by pulse vortexing, and 5 µL of each
filtered dry air, and then treated with oxygen plasma (ION3, Corona, CA) sample was then placed on a slide, heat fixed, and dyed with a
(100 mW, 1% oxygen) for 1.5 min to form radical groups. To generate Christmas Tree stain as a confirmatory test before applying samples
thiol groups, the slides were placed into a 4% v/v solution of 3-MPS into the microchannels.[44]
in absolute EtOH and incubated for 30 min at room temperature. Sperm Lysis On-Chip: To lyse sperm cells and collect DNA on-chip,
After the silanization step, the surfaces were rinsed with EtOH to TCEP was utilized as a lysis agent, and 20 µL of TCEP solution were
remove unbound chemical residues and dried using either N2 gas or introduced (20 µL of TCEP + 1980 µL of RNase free water + 20 µL of
filtered dry air. After the microfluidic chip’s three components were Triton X-100 (100%), pH was adjusted to pH 2.5 with HCl) into the
assembled, GMBS (10 × 10−3 m in DMSO:PBS (1:1)) was introduced microchannel, and then, incubated for 15 min. An additional 80 µL of
into the microchannels to form succinimide groups by incubating for TCEP solution was applied into the channel, and the lysate was collected
45 min at room temperature. The microchannels were then washed in an eppendorf tube.
with 1xPBS (40 µL, 2 times). 4-ABAH reagent (0.25 and 2 mg mL−1 After completion of cell lysis in all experimental sets, 40 µL of
in 1:1 (v:v) ratio of DMSO:1xPBS) was utilized to form hydrazide Proteinase K solution (1 µg mL−1) were added to each lysate tube
groups for immobilization of SLeX molecules to the microchannels and incubated for 4 h at 55 °C. During incubation, the tube inverted
surface. After a washing step with 1xPBS (40 µL, 2 times), different occasionally to disperse the sample. Followed by the incubation, 100 µL
concentrations of SLeX ranging from 0.1 to 0.5 mg mL−1 were of Buffer AL and 100 µL of ethanol were added to the samples and mixed
prepared using the stock SLeX solution (1 mg mL−1) and applied to by vortexing. The samples were then run through gDNA extraction using
the microchannels. These surfaces were incubated overnight at +4 °C. a Qiagen spin column protocol.
The microchannels were then washed with 1xPBS (40 µL, 2 times) and Qiagen Spin Column Protocol: All samples were processed through
the surface functionalization was accomplished with BSA (3% (w:v) in the spin column procedure according to the manufacturer’s protocol.
1xPBS) incubation for an hour at room temperature to minimize/avoid The samples were applied to the QIAamp Mini spin column in a 2 mL
nonspecific binding. collection tube without wetting the rim. The tubes were centrifuged at

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6000 × g (8000 rpm) for 1 min. The QIAamp Mini spin column was Keywords
placed in a clean 2 mL collection tube and the tube containing the filtrate
was discarded. 500 µL of Buffer AW1 was then added without wetting bioinspired materials, differential extraction, DNA casework backlog,
the rim. The tubes were again centrifuged at 6000 × g (8000 rpm) for forensic cases, microfluidics
1 min. After that, the QIAamp Mini spin column was placed in a clean
2 mL collection tube, and the collection tube containing the filtrate was Received: January 22, 2018
discarded. 500 µL of Buffer AW2 was added without wetting the rim Revised: April 13, 2018
and centrifuged at full speed (20 000 × g; 14 000 rpm) for 3 min and Published online: June 15, 2018
the old collection tube with the filtrate was discarded. The tubes were
again centrifuged at full speed for 1 min. The QIAamp Mini spin column
was placed in a clean 1.5 mL microcentrifuge tube and the collection
tube containing the filtrate was discarded. 50 µL of Buffer AE or distilled
water was added. The tubes were then incubated at room temperature [1] The Rape Abuse & Incest National Network(RAINN) Victims of
for 1 min and centrifuged at 6000 × g (8000 rpm) for 1 min. This step Sexual Violence: Statistics, https://www.rainn.org/statistics/vic-
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sample. [2] L. A. Greenfeld, Department of Justice, Office of Justice Programs,
Quantitation of Extracted DNA: DNA concentration of each sample Bureau of Justice Statistics, Sex Offenses and Offenders, 1997, https://
was quantified using the Qubit Fluorometer. The manufacturer’s bjs.gov/content/pub/pdf/SOO.PDF.
protocol for Qubit dsDNA High Sensitivity (HS) Assay (Thermo [3] R. E. Morgan, B. J. Mason, Department of Justice, Office of Jus-
Fisher Scientific, Waltham MA) was followed. First, the Qubit working tice Programs, Bureau of Justice Statistics, Crimes Against the Elderly
solution was prepared by diluting the Qubit dsDNA HS Reagent 1:200 (2003–2013), 2014, https://www.bjs.gov/content/pub/pdf/cae0313.
in Qubit dsDNA HS Buffer. Then, two standards were prepared by pdf.
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After that, sample solutions were prepared by adding 2 µL of each eral money, USA Today, http://www.usatoday.com/story/
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